The purpose of this Shared Instrumentation Grant application is to obtain a TIRF/widefield fluorescence microscope, the first in North Dakota, to enhance existing NIH-funded research areas and to develop new avenues of research. The long-term goals of the projects requiring this instrumentation are to understand the mechanisms underlying the pathogenesis of several disease states under investigation at the University of North Dakota including Alzheimers? disease, Parkinson?s disease, inclusion body myositis (IBM), epilepsy and many more. The overall aims of these projects are to 1) investigate the role of endosomal/lysosomal function in amyloidogenesis associated with Alzheimer's disease, 2) investigate the role of TRPC calcium channels and dysregulation of calcium homeostasis in dopaminergic neuronal cell death associated with Parkinson?s disease and salivary gland dysfunction, 3) investigate the role of palmitoylation and phosphorylation in regulating dopamine transporter trafficking and function, and 4) investigate mechanisms underlaying cholesterol mediated neurodegeneration associated with Alzheimer's disease. Both TIRF microscopy, an imaging technology that allows very precise realtime fluorescent imaging of dynamic molecular events at the cell membrane surface, and state of the art widefield fluorescence microscopy are necessary for investigators of the proposed projects to extend their studies. All of the projects are investigating dynamic molecular events within different cellular compartments. TIRF microscopy is essential to meeting the goals of several of these projects because of their focus on molecular processes at the cell membrane or the need for single molecular fluorescence imaging. In addition, all projects require fluorescence microscopy instrumentation configured for quantitative wide field fluorescence imaging of dynamic events over time using multiple channels and multiple sampling locations to provide statistical rigor. These studies cannot be accomplished with existing fluorescence and confocal imaging equipment. The closest TIRF/wide field fluorescent microscope systems to the University of North Dakota are at the University of Manitoba (160 miles) and the University of Minnesota (350 miles). These distances (and an international border), our documented needs, and the inability of others to accommodate our needs underscore the acute need for this equipment. To achieve our research goals, we propose to purchase and operate an Olympus IX81-ZDC2 cellTIRF microscope system. This microscope will be integrated into our already functional imaging core facility in association with other imaging equipment and will take advantage of existing web-based scheduling, technical support, web-based data storage and sharing, and system contract support. Acquisition of this instrumentation will build institutionally on our ver successful COBRE and INBRE models. The research enterprise at the University of North Dakota has grown substantially to almost $130 million in 2011 with recent infrastructure improvements including vast expansions to the School of Medicine and Health Sciences, a neuroscience research building, and new laboratories for micro- PET, confocal, electron and stereological imaging, mass spectrometry, electrophysiology, as well as animal work involving transgenic and gene knockout technologies, infectious agents and radioactivity, and behavior. This TIRF/wide field fluorescence microscope system will advance the basic science research of our faculty members in a wide range of fields and will lead to development of new research areas.
|Schott, Micah B; Gonowolo, Faith; Maliske, Benjamin et al. (2016) FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca(2+)/PKA crosstalk. Cell Signal 28:294-306|